This proposal is a cont1nuing effort to elucidate the molecular mechanism of the human erythrocyte glucose transporter function. The long term objects are to understand the nature of the defect underlying insulin-resistive states seen in Type II diabetes and obesity. More immediate objectives are to determine tertiary structure of the transporter and the conformational dynamics associated with the transport function. The amino acid sequence of this protein is known, which predicts that the protein is made of twelve transmembrane segments and four nontransmembrane hydrophilic segments. Based on these predictions, the following experiments are proposed. First, these transmembrane and nontransmembrane segments will be isolated and identified by protease-digestion. HPLC-separation and determination of characterlstic amino acid contents predicted by the model. The three dimensional arrangement of these segments will be determined by labelling them with crosslinking reagents, lipophilic covalent probes, and tritiated water protons. Possible changes in these three dimensional arrangements upon the removal of the nontransmembrane, hydrophilic segments will be monitored by the same procedures. The changes will also be monitored based on circula, dichroism, Fourier transform infrared spectroscopy, radiation inactivation target size and fluorescence energy transfer measurements. The substrate binding sites in the substrate recognition pockets and in the translocatlon pathway in the protein will be studied by tagging them with photoreactive substrate analogs. Side-specific, nontransportable analogs and transportable analogs will be used to differentiate these sites. Cytochalasin B binding pocket will be characterized by identifying its binding site. Those transmembrane or nontransmembrane hydrophilic segments that are affected by the substrates or inhibitor-induced conformational perturbation will be determined by labelling and identifying the amino acid residues whose reaction to certain alkylating reagents are known to inactivate the protein function and are sensitive to the substrate and inhibitors. Four different alkylating reagents will be used for this purpose. Lastly, as a long term goal, crystallization of the purified transporter protein will be attempted for future x-ray diffraction studies.
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